Method and device for quantitative end point determination in immunofluorescence using microfluorophotometry

ABSTRACT

The present invention discloses a device and a process for quantitation of end point in the formation of fluorescent reaction product in microfluorophotometry. The process comprises: 
     (a) incorporating a protective agent in a suitable mounting medium in an amount sufficient to reduce fading of fluorescent reaction product less than 25% of initial fluorescent intensity; 
     (b) calibrating photometer used in said microscopy with a stable emitter; and 
     (c) recording the intensity of fluorescence of said fluorescent reaction product by means for measuring light intensity. 
     The invention also includes a device for calibration of the photometer and a kit comprising separate containers for suitable mounting medium, buffer, suitable immunofluorescent reagents, fading retardant means, a photometer calibrating device and the like and optional instructions.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention is related to a method and device for quantitativedetermination of fluorescent reaction product endpoint inimmunofluorescence using microfluorophotometry. More particularly, thepresent invention is related to standardization of various parameters,calibration of components or devices and determination of reactionconditions for a reliable, stable and reproducible quantitation offluorescent reaction product in immunofluorescent microscopy ormicrofluorophotometry.

2. Prior Art

Current method for immunofluorescent (IF) test involves a subjectiveevaluation of the end point (titer) which is dependent, inter alia, uponthe observer's expertise, experience and judgement. This subjectivity isfurther complicated by the rapid fading of the fluorescent reactionproduct (FRP) under the test conditions routinely employed. Thus, as theart is presently known, the outcome of an IF test becomes a function oftime and judgment. In fact, there being no better or objective methodfor IF assay, researchers have generally conceded that rapid fading offluorophores would have to be tolerated if IF is opted as the procedureof choice. Nairn et al., Clin. Exp. Immunol. 4, 697-705 (1969); Johnsonet al., J. Immunol. Meth. 55, 231-242 (1982); Schauenstein et al., J.Immunol. Meth. 8, 9-16 (1975); Wick et al., Ann. New York Acad. Sci.254, 172-174 (1975); McKay et al., Immunology 43, 591-602 (1981).

Various techniques have been used to protect the sample from fading.These are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        METHODS FOR THE REDUCTION OF FADING                                           Technique       Investigator                                                  ______________________________________                                        (1) Localization under phase                                                                      Ploem, Golden, Fukuda, Geyer                                  contrast                                                                  (2) Fast, epi-shutter                                                                             Ploem, Golden, Geyer, Kaufmann                                excitation      Nairn                                                     (3) Chemical Agents Gill, Johnson, Sedat, Giloh,                                                  Kaplan, Picciolo                                          (4) Pre- or Post-illumination                                                                     Fukuda, Fujita                                            (5) Variable iris diaphragm                                                                       Goldman, Ploem                                                on objective                                                              (6) Neutral density filters                                                                       Nairn, Ploem                                              (7) Light sources   Goldman, Haaijman, Johnson                                (8) Excitor/barrier filters                                                                       Goldman, Haaijman, Nairn,                                                     McKay                                                     (9) Field diaphragms                                                                              Golden, Ploem, Haaijman                                   ______________________________________                                    

Of these, a more practical and feasible technique appears to be the useof chemical additives in the mounting medium to protect the fluorophorefrom the effects of the excitation light.

Protection from fading would make exposure of the specimen to theexciting light less critical. This would allow ease in the localizationof the fluorescent specimens and permit more accurate discriminationbetween weakly positive and negative results, which is difficult if thesample is rapidly fading.

Certain tests, such as determination of the type of Herpes present,require finding any positive cells that may be present on the entireslide. This searching procedure may take several minutes and must bedone during excitation to recognize the presence of the positives. Iffading is rapid, positives may be missed. Protection from fading inthese cases is critical.

Reducing the fading would also significantly improve quantitation of theFRP on the IF microscopy slides. Retarding fading would permit longerscan times on slides without concomitant decrease in fluorescenceintensity. This would permit the use of automated or semi-automatedinstrumentation which could scan a slide and determine the endpointquantitatively.

Rapidly fading specimens account for many false negatives in theclinical laboratory. In some cases, by the time the technician has setup the slide on the microscope, the weakly positive cell has faded to anegative cell. In the case of antinuclear antibody (ANA) positive cells,the technician cannot properly identify the staining pattern if thespecimen is rapidly fading. Stabilization of the fluorescence emissionis, therefore, necessary for objective and quantitative determination ofantibody level.

A factor which must also be considered in the evaluation of the IFassay, is the variability due to the instrumentation.

Earlier methods used microcapillaries filled with the fluorophore ofinterest. In this method the microcapillary diameter is measuredmicrointerferometrically, allowing exact calibration of the microscopefluorometer and correlation of the measured fluorescence intensitieswith the mass of the excited fluorochrome. Serntz, et al., FluorescenceTechniques in Cell Biology Springer-Verlag, N.Y., pp 41-49 (1973).Certain applications may not require an exact calibration, but aneasier-to-perform secondary standardization may be satisfactory.

Recent work by Jensen et al., J. Immuno. Meth. 42: 343-353 (1981) used aparticle of europium salt phosphor as a daily standard.

The Applicants have now improved the immunofluorescent methodologies byvarious means amongst which are: (a) removing the subjective evaluationof endpoint determination by providing a quantitative measure; (b)providing a reagent-reactivity-monitor which includes performancetesting associated with a numerical value, (c) providing a calibrationstandard for interlaboratory brightness comparison; and (d) achieving aquantitative measure by a fading retardant means.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide an agentto reduce fading of fluorescent reaction product in amicrofluorophotometric test system.

It is another object of the present invention to provide a reproduciblystable system for quantitative determination of fluorescent reactionproduct in microfluorophotometry.

It is a further object of the present invention to provide a device andmethod for use in a photofluorescence system to measure microscopicallyobserved epi-fluorescence intensity in terms of electromotive force.

It is yet another object of the present invention to provide a methodusing a computer-controlled microscope-photometer for measuringfluorescence intensity as a voltage output.

It is still another object of the present invention to providecalibrators for inter- and intra-laboratory comparison of fluorescencemicrophotometry.

These and other objects and advantages will become evident as thedescription of the invention proceeds.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantagesof the invention will be better understood upon a reading of thefollowing detailed description when considered in connection with theaccompanying drawings wherein:

FIG. 1 is a schematic of light path for the Zonax microscopefluorophotometer wherein the epi-fluorescence exciting light (mixed)transmitted visible light (small dash) and the emission light (longdash) paths are shown.

FIG. 2 shows three dimensional graph of the signal to noise ratio versusilluminating field area and PMT aperature area of a fragment of NBSthread.

FIG. 3 shows correlation of the corrected fluorescent intensity readingsof the thread with high voltage settings.

FIG. 4 shows fluorescent intensity of Corning plate versus burn hours ofone 100 W mercury lamp over a period of three months.

FIG. 5 shows scatter plot of fluorescent intensity versus distance movedin microns across Corning plate showing linear regression line.

FIG. 6 shows stability of phosphor particles in various mounting mediaunder continuous excitation.

FIG. 7 shows comparison of measured intensity of fluorospheres withbrightness based on dye concentration.

FIG. 8 shows the relationship of optimization of reducing agentconcentration for protection from fading of FITC-labelled antibodiesafter 30 min excitation, the % remaining intensity in the Perkin-Elmeris plotted vs the reducing agent concentration in moles/liter. DABCO wastested on Rubella conjugate (open squares) and the DTT was tested onToxoplasma gondii conjugate (open circles).

FIG. 9 shows the relationship of fading of cells on test kit slides withand without 0.025M DTE added to buffered glycerol mounting medium, the %remaining intensity is plotted vs time in min for Toxoplasma (squares),Rubella (triangles), and ANA (circles) with DTE (open) and without anyreducing agent (solid).

FIG. 10 shows the relationship of the effect of reducing agent on thefading of FITC-labelled ANA cells with rhodamine counterstain duringcontinuous excitation, the % remaining intensity vs the time in min ofexcitation is plotted. The ANA cells were mounted in buffered glycerol(triangles) and with 0.025M DTE (squares) or 0.3M DABCO (circles).

FIG. 11 shows the effects of DTE concentration on fading ofFITC-labelled ANA cells with rhodamine counterstain during continuousexcitation, the % remaining intensity is plotted vs the time in min ofexcitation for cells with 0.025M (squares), 0.033M (circles) and 0.025M(triangles) DTE added to the buffered glycerol.

FIG. 12 shows the best fit regression curves for fading of FITC-labelledRubella cells during continuous excitation when mounted in bufferedglycerol (open circles) or 0.05M DTE (solid circles).

FIG. 13 shows the improvements in fluorescence conversions in accordancewith the present invention. Proposed fluorescence conversions showingrelative energy levels: Excitation light excites ground statefluorophore (F₁ -S₀) to excited singlet (F₁ -T₁). Light emission occurswith decay to ground state or intersystem crossing to triplet (F₁ -T₁)and then decay to ground state. Radiationless decay by interaction withoxygen to oxidized fluorophore (F₁ -O) is inhibited in the presence ofreducing agents.

DETAILED DESCRIPTION OF THE INVENTION

Various objects and advantages as suggested herein are achieved by thepresent invention which includes a method for quantitative determinationof fluorescent endpoint in fluorescent microscopy comprising:

(a) incorporating a protective agent in a suitable mounting medium in anamount sufficient to reduce fading of fluorescent reaction product lessthan 25% of initial fluorescent intensity;

(b) calibrating photometer used in said microscopy with a stableemitter; and

(c) recording the intensity of fluorescence of said fluorescent reactionproduct by means for measuring light intensity.

As used herein fading refers to the time-dependent decrease influorescence intensity upon continuous exposure to ultraviolet (UV)exciting light, as distinguished from quenching, which is a staticreduction in the intensity due to some environmental or chemicalcondition present. Changes in the fluorescence intensity with time are ameasure of the fading rate, while differences in the initial intensityunder various environmental conditions are an evaluation of quenching.

A. Instrumentation

Any instrument suitable for use in fluorophotometry can be used. Twogeneral classes of instrumentation which could be employed for intensitymeasurements of FRP are as follows.

I. Macrofluorophotometers

Macrofluorophotometers are designed to accept a cuvette that holds avolume of fluorescent solution or a solid sample holder whichaccommodates a flat plate. Both an excitation and an emissionmonochromator with variable slit widths are in the optical path (Sernetzand Thaer, Fluorescence Techniques In Cell Biology. New York:Springer-Verlag, pp 41-49, 1973). A corrected emission spectrum isobtained using a standard emittor as a wave length calibration.Macrofluorophotometers are effectively used for screening of the effectsof the chemical environment on the fluorescence emission intensity,monitoring the purification procedures for the production of labeledconjugate and determination of the excitation and emission spectral offluorophores. Detailed description of a Perkin-Elmer 650-40spectrofluorophotometer and its use in predicting effectiveness ofreducing agents to protect the FITC-labeled conjugates from fading isprovided herein infra. Only part of the solution of the fluorophore in acuvette in a macrofluorophotometer is exposed to the excitation beam.The rest of the molecules are free to diffuse and effectively replenishthe faded molecules, particularly if the solution is being mixed.Therefore, confirmation of the effectiveness of fading protection mustbe made in the microfluorophotometers.

II. Microfluorophotometers

Various types of microscopes are available with epi-fluorescenceexcitation and with a photometer to detect the emitted light intensityand convert it to a digital signal and any one system could be used.Some of such systems have been described by Ploem, Chap. 10:Quantitative Immunofluorescence, pp. 63-73, Blackwell Sci. Pub. (1970);J. Histochem. Cytochem. (22), 7, 668-677 (1974); Ann. New York Acad.Sci. Vol. 254, pp. 4-20 (1975); Chapter 6: Automated Methods inImmunofluorescence Studies, pp. 73-94 (1982); Taylor and Heimer, J.Biol. Standardization 2, 11-20 (1974); Thaer, Acad. Press, New York,Vol. 1 pp. 409-426 (1966); Golden and West, J. Histochem. Cytochem.22(7), 495-505 (1974).

FIG. 1 shows a typical epi-fluorescence optical path whereby theexciting light is incident through the objective on the specimen and thefluorescence emission is collected by the same objective and thenfocused on the photocathode surface of the photomultiplier tube (PMT).An advantage of epi-illumination is a reduction of distortion of theemission spectrum due to reabsorption where there is an independence ofsection thickness. Therefore, one can measure surface fluorescene ofopaque objects. There is also, more precise alignment due tosimultaneous focusing since the objective is the condensor (Rigler, ActaPhysiolog. Scandin. 67 (supplementum 267), pp. 117 (1966); Pearse andRost, J. Microsc. 89(3), 321-328). Several microscope systems arecommercially available with microprocessor control, including theoperation of a 0.25 or 0.5 micron scanning stage. A preferred ZeissZonax system used herein is described infra.

B. Fluorescence Affecting Factors

In order to appreciate the invention, several factors which affectfluorescence intensity should be understood. These are summarized below.

I. Optical

Some of the optical components which can contribute to fading are asfollows.

1. lamp housing

Depending on the design of the housing for the excitation light source,the amount of light reflected to the collector lens will vary. Since thefading is dependent on the excitation energy, if the excitation light isscattered in the lamp housing and is lost through the baffles, this isequivalent to a decrease in excitation energy and will result in lessfading and less signal. The better designed lamp housings reflect a highpercentage of the excitation light on the collector lens. In addition,lamp housing vary in their efficiency of dissipating the heat from thelight source. Heat build-up can cause instability of the light source,i.e. wandering of the arc or misfiring of the arc. An unstable lightsource causes fluctuation in the output from the lamp and will causevariations in the emission.

2. light source

There are a variety of light sources available for fluorescenceexcitation. Lasers offer the advantage of delivering monochromatic lightand can generate pulses of light as short as 0.4 microseconds, Wick etal., Ann. New York Acad. Sci. 254, 172-174 (1975); Bergquist Scand. J.Immunol. 2, 37-44 (1973). These sources also give a high output ofenergy for exciting weakly fluorescent specimens. Additionally, lasershave a long lifetime compared to conventional light sources such asmercury, xenon or halogen lamps. Mercury arc lamps emit strongly atseveral lines in the UV and blue light regions (365 nm, 405 nm and 435nm). Even though there is no special line in the spectral range offluorescein isothiocyanate (FITC) absorption (440-490 nm), these sourcesare good for FITC emission (Goldman, Acad. Press, N.Y. (1968); HaaijmanInst. Experim. Gerontol. (1977). Xenon bulbs produce a continuousemission throughout the entire spectrum but the brightness per unit areais lower than with mercury bulbs (Goldman, Acad. Press, N.Y. (1968). Inaddition, xenon lamps require the use of more restrictive filters thanwith mercury bulbs, since the excitation light continues into theemission region of the dye (due to the continuous spectrum). Xenon andmercury bulbs cause considerable fading of the specimen. Halogen lampsdo not emit as much blue light and emit lower intensities than mercurylamps. Therefore, halogen lamps, in general, are not suitable forfluorescence quantitation (Goldman, Acad. Press, N.Y. (1968). Theselamps are useful when the specimen is brightly stained and the observerwants to eliminate fading as much as possible. Recently, the HBO 100 Wmercury lamps have been developed with more stable arcs, more excitationenergy and less heat output energy. They are operated with a stabilizedpower supply and are preferable.

3. Excitation energy

Ploem, Ann. New York Acad. Sci. 177, 414-429 (1971) has shown that thefading rate is dependent on the excitation energy. Most researchers whoperform experiments to measure fading have not measured the excitationenergy of the light source, because the instrumentation is specializedand expensive. The output varies from day to day and decreases as thebulb ages. Factors such as type of light source, age of bulb, positionof the collector lens, diffusion of the light beam over the specimen,type of heat filters, magnification and numerical aperture (NA) of theobjective and the excitation filters all affect the excitation energy.It is clear, therefore, that the fading of the fluorescently stainedspecimens reported in the literature cannot be compared unless relatedto the power density of the excitation light source.

4. Collector lens

As the light exits the lamp housing, the collector lens concentrates ordiffuses the light. The excitation energy is dependent on the positionof the collector lens. If the collector lens is adjusted so that thelight is focused on a small spot on the specimen, then the energy perunit area will be higher than if the light is diffused over the entirefield. Therefore, if the light is concentrated rather than diffused inorder to increase the emission intensity, there will be increased fadingof the specimen.

5. heat filters

Heat filters are placed in the light path to filter out the infra-redradiation so that the excitation filters are not cracked by theconstant, intense radiation from the light source. In addition, thesefilters will decrease the transmission of light in the UV region tovarying degrees, depending on the type and quality of the filter.Goldman, Acad. Press, New York (1968).

6. excitation and neutral density filters

The amount and wavelength of the exciting light reaching the specimen isdependent on the filters used. Broad-band excitor filters allow widerwavelength band to reach the specimen with more fading than narrow-bandexcitor filters. McKay et al., Immunology 43, 5910602 (1981) showed thatusing narrow band FITC filters for blue light instead of Uv+blue,reduced the fading and fluorescence intensity by equal amounts. Herzoget al., J. Immunol. Meth. 3, 211-220 (1973) also found that the rate offading is dependent on the filters used. Schauenstein et al.,"Immunofluorescence and Related Staining Techniques" pp. 81-95 (1978)compared the excitation spectra of free FITC and conjugated FITC. Theyfound that conjugation of protein to the FITC molecule quenches the UVmaxima at 280 and 340 nm (the UV region) as compared to free FITC. Sincethere is no quenching at the 496 peak (the blue region), blue excitationis preferrable when high intensities are desired. Ploem, Ann. New YorkAcad. Sci. 177, 414-429 (1971) compared the fading of antinuclearantibody (ANA) positive cells stained with FITC using various excitationfilter combinations. The first combination (GG 475 and two KP 490filters) which has a high transmittance (about 80%) showed a very rapidloss of intensity within 0.25 second. The second filter combination (thefirst with a 25% transmittance neutral density filter added) showed amuch slower decay of the fluorescence intensity. Enerback and Johansson,Histochem. J. 5, 351-362 (1973) showed that inserting graded neutraldensity filters into the exciting light path proportionally reduced thefading. Dichroic mirrors are interference dividing plates that reflectlight of certain wavelengths through the objective and allow light ofshorter or longer wavelengths to pass through the filter, being lostthrough scattering. Ploem Chapter 10: Quantitative Immunofluorescence,pp. 63-73 (1970). The fading could be significantly enhanced or reduceddepending on how selectively the dichroic mirrors are filtering out thelight.

7. objectives

Since in epi-fluorescence, the objective acts as a condensor, theintensity of the light is dependent on the numerical aperture (NA) ofthe objective; the intensity increases as the square of the NA (Goldman,Acad. Press, New York (1968); Haaijman, Inst. Experim. Gerontol. (1977).The NA is defined as the product of the refractive index of the mediumin which the aperture angle is measured and the sine of the apertureangle, Piller Springer-Verlag, New York (1977). A typical NA for lowpower objective is 0.65 and 1.25 for high power objective. Someobjectives have a variable iris diaphragm which allows the control ofthe excitation of the specimen. While reducing the excitation (via thismethod) does reduce fading, it does not allow absolute quantitation ofthe emitted intensity. Unless there is a very specific way of assuringthat the iris diaphragm is set to exactly the same place each time, onecannot absoutely compare the intensities of samples. The type ofobjective will also influence the fading. If the objective is made ofseveral lenses which have been cemented together, there is approximatelya 4% light loss each time the light passes through an air-glassinterface, Zeiss (1960). Depending on the number of lenses in theobjective, this light loss could be significant if one is attempting toquantitate the fluorescence intensity. It should be noted that a similarlight loss is observed in excitation filters which are composed ofseveral filters cemented together.

II. Excitation time

Not only is fading dependent on the optical factors but also onexcitation energy and the time and period of exposure. The longer afluorescently-tagged specimen is exposed to the exciting light, the morethe fading will occur, down to a minimal plateau level. Interspersingexcitation with dark periods has an effect on the fading in some cases.This may result in recovery of some of the intensity and is variablewith fluorophore, exposure and dark times, and excitation energy.

III. Environmental

In addition to the fading caused by the optical elements and excitationtime, there are environmental factors which may affect fading. HaaijmanInst. Experim. Gerontol. (1977) compared the fading ofaminoethyl-Sephadex bound FITC and Sepharose-bound FITC for 2 min undercontinuous excitation. FITC coupled to Sepharose faded 20% more thanFITC coupled to aminoethyl Sephadex beads. It is concluded that fadingis dependent on the matrix to which FITC is bound.

Haaijman, supra, tested the influence of pH on the fading in thepresence or absence of protein (i.e. CNBr activated4B-Sepharose-OVA-FITC vs 4B-Sepharose-FITC) to test the hypothesis thatelectrophilic groups near the FITC moiety influence the fading. Sincethe fading in the presence or absence of protein was similar at variouspH levels, it was concluded that fading is not influenced byelectrophilic centers in the protein to which it is coupled, but is aproperty of the molecule itself. McKay et al., Immunology 43, 591-602(1981) found that when the pH of the buffered blycerol mounting mediumwas raised from 7.2 to 8.8, there was a 23% increase in the fluorescenceintensity, but the rate of fading did not change.

Comparison of Fading

Since several factors, as enumerated above, affect the intensity of theemitted light, a comparative study of fading performed in variousoptical set-ups in different laboratories is difficult to make. No setfading parameters have been established to allow this comparison.Therefore, the fading percentages obtained at different times fromvarious investigators are not necessarily comparable. However, anappreciation of the relative effectiveness of various conditions can beobtained from the following description.

A. Excitation Source 1. Laser

Several authors have used lasers to measure the fading of FITC-labeledconjugates (Wick et al., Ann. New York Acad. Sci. 254, 172-174 (1975);Kaufman et al., J. Histochem. Cytochem. 19, 469 (1971); Bergquist,Scand. J. Immunol. 2, 374-44 (1973); Bergquist et al., Ann. New YorkAcad. Sci. 254, 157-162 (1975); Schauenstein et al., J. Histochem.Cytochem. 28(9), 1029-1031 (1980). These investigators have compared thefading of the conjugate when the sample was excited by repeated, shortpulses of light with a laser to the fading when the sample was exposedto a conventional light source such as a mercury or xenon arc lamp.Additionally, lasers have been used to measure recovery (percentage ofthe initial fluorescence intensity that is regained as the cells areleft in a dark environment) following various periods of fluorescenceexcitation. Experiments combining fading and recovery have been usefulin explaining the mechanism of fluorescence fading.

a. Argon-ion. laser

Kaufman et al., J. Histochem. Cytochem. 19, 469 (1971) measured thefading of FITC-labeled Escherichia coli cells using an Argon-ion lasersource. They found that when using an Argon laser at a power density of160 watts/cm², 89% of the initial E. coli intensity had faded within 10seconds, under continuous irradiation. But, when the excitation time wasreduced to milliseconds, no significant fading could be detected.Schauenstein et al., Immunofluorescent Technology, pp. 27-36 (1982)found that free FITC in solution lost 40% of its initial intensityduring the first 100 milliseconds of excitation with an Argon laser.

b. Pulsed dye laser

Bergquist and Nilsson, Ann. New York Acad. Sci. 254, 157-162 (1975)compared the fading of FITC-labeled glutaraldehyde-polymerizedmicrospheres of purified human IgG excited with an HBO 200 W mercurylamp to the fading when the spheres were excited with a Chromabeam 1070pulsed dye laser. The laser was adjusted to produce light of 495 nm.Bergquist, Scand. J. Immunol. 2, 37-44 (1973) has previously shown thatwhen the spheres were exposed to a total of 125 pulses (each pulse is0.4 μs for a total exposure of 50 μs) and the resultant image wasexposed to photographic emulsion, there were no signs of significantfading. In a second study (Bergquist and Nilsson, Ann. New York Acad.Sci. 254, 157-162 (1975), they repeated the previous study andquantitatively measured the fading by monitoring the deflections on anoscilloscope from the photomultiplier tube (PMT). They found that evenafter 50 laser pulses had illuminated an individual sphere, no fadingwas observed. However, after one second of exposure to a HBO 200 Wmercury light, only 85% of the initial intensity remained.

c. Recovery

Since researchers and lab technicians are usually interested inobserving a fluorescently stained field more than once (i.e. in thehistopathological diagnosis of cancer cells or when observing thestaining pattern in immunofluorescence diagnostic test kits),researchers are interested in determining the extent of permanentlowering of the intensity by prior excitation conditions. Maintainingthe level of the initial intensity is important in the diagnosis ofdisease states since it is often necessary to have a second technicianor a physician review the test results. If the fluorescent field wasirreversibly faded during the initial observation, then it is notpossible to confirm the first technician's diagnosis and false positiveor false negative results may be reported. Kaufman et al., J. HistochemCytochem. 19, 469 (1971); Wick et al., Ann New York Acad. Sci. 254,172-174 (1975) and Schauenstein et al., J. Immunol. Meth. 8, 9-16 (1975)found that recovery is dependent on the time of exposure to theexcitation light souce and the length of time the specimen is left inthe dark following excitation. These authors found that minimum darkperiod of two seconds between laser pulses is necessry to get recoveryof the fluorescence. Schauenstein et al., "ImmunofluorescenceTechnology" pp. 27-36 (1982) found a 60% recovery using two pulses of 3ms with a 3 ms (millisecond) dark interval. However, in many cases,recovery does not occur or only a partial recovery of the initialintensity occurs. Recovery is negatively related to the product of theexcitation time and intensity of the exciting light and is positivelyrelated to the time the sample is left in the dark following excitation(Wick et al., Ann New York Acad. Sci. 254, 172-174 (1975); Kaufman etal., J. Histochem Cytochem. 19, 469 (1971); and Schauenstein et al., J.Immunol. Meth. 8, 9-16 (1975).

2. Conventional light sources

Most fluorescence microscopes, i.e., those in hospital laboratories orthose used for research, are equipped with either a halogen, a mercuryor a xenon light source. The average laboratory can neither afford alaser excitation source nor has the personnel qualified to properlyalign the light source. In addition, laser light sources require thatspecial low-fluorescence optics and filters be used to avoidautofluorescence of the optical system.

a. Mercury lamps

Nairn et al., Clin. Exp. Immunol. 4, 697-705 (1969) measured the fadingof rat gastric cells stained with FITC-conjugated anti-human globulinusing a HBO 200 W mercury lamp. When the specimen was mounted inbuffered glycerol at pH 8.6 and excited with only ultraviolet light(UV), 35 s (seconds) were required to fade half the initial intensity.When the sample was excited with UV+blue light, the half-life decreasedto four seconds. After one minute continuous excitation with UVirradiation, only 30% of the initial intensity remained. Haaijman Inst.Experim. Gerontol. (1977) compared the fading of aminoethyl-Sephadexbound tetramethyl rhodamine isothiocyanate (TRITC) and membrane-boundTRITC. Membrane bound TRITC faded about 25% more in 2 minutes than theTRITC bound to the beads. He found the FITC and TRITC bound toaminoethyl sephadex beads faded less rapidly than cell-bound conjugates.Golden and West J. Histochem. Cytochem. 22(7), 495-505 (1974) measuredthe fading of Ehrlich's hyperdiploid mouse ascites tumor cells stainedwith acridine orange using a HBO 100 W mercury lamp. They described thefading in terms of a time constant, tau, which is approximately 1.8 sec.Although not specifically stated, it can be inferred from the fadingcurve that tau is the time required to fade to 37% of the initialintensity. The data showed that the fading can be approximated with asingle exponential. The shape of this fading curve was dependent on thecell type and the substrate biopolymer.

b. Xenon lamps

Using a XBO 75 W xenon lamp, McKay et al., Immunology 43, 591-602 (1981)measured the fading of conjugates of anti-human gamma globulin-FITC andanti-human gamma globulin-rhodamine B 200 (RB 200). With the RB 200conjugates, there was little, if any, fading after two min, and thisdecline could not be separated from instrument error. For fluorescein,however, there was considerable fading which reached a plateau after acertain period of time. This result was interpreted to mean that fadingis the sum of two components, one that decays exponentially and one thatremains constant. They subtracted the plateau level value, whichrepresents the non-fading component, from each intensity value andplotted the fading component vs time on semi-logarithmic paper. Thisplot produced a straight line which showed the fading obeyed first-orderkinetics. They found a half-life of about 1 min for their FITCconjugates. Enerback and Johansson Histochem. J. 5, 351-362 (1973)measured the fading of several fluorochromes including FITC andFeulgen-Schiff using a XBO 75 W xenon lamp and instrumentation capableof recording fluorescence of very short duration. They found a half-lifeof two 2 seconds for FITC under continuous excitation. ForFeulgen-Pararosaniline reaction, there was a 20% loss of initialfluorescence after 20 seconds. They also tested the effect of repeatedvery short excitation times at two second intervals on the fading. ForFITC, there was significant fading after fifteen measurements withillumination times up to 1/60 second. Using an oscilloscope, they founda fading of 0.5% during the first two 2 ms of illumination. ForFeulgen-Schiff stained cells, the fading could be prevented by reducingthe illumination time. Bohm and Sprenger Histochemie 16, 100-118 (1968)measured the fading of sperm stained with several dyes, includingAcriflavin and Pararosaniline under 5 min continuous excitation using aXBO 150 W xenon lamp. They found a fading rate of 25% and 60%,respectively, for Pararosaniline and Acriflavin.

c. Recovery

McKay et al., Immunology 43, 591-602 (1981) tested the recovery ofFITC-stained cells using a 75 W xenon bulb. They allowed the FITCconjugate to fade approximately 6 half-lives and then measured theintensity by varying the dark period between excitations using anexcitation shutter. They found that if the shutter was opened for onlythree seconds every five min., the intensity increased from 64 to 99(33%). This recovery appeared real because it could not be obtained onunstained specimens. This data correlated with the recovery experimentsperformed using lasers.

Methods of Protection

In light of the above, the Applicants reached the conclusion that inorder to improve the accuracy of the IF test and to achieve quantitationof the fluorescently emitted light, it is essential at least tostabilize the fluorescence emission. The Applicants, therefore, deviseda chemical system to accomplish this result. The Applicants have alsodiscovered that when selecting possible chemical agents to retardfading, it is important that the agents do not fluorescence at or nearthe excitation or emission wavelengths of the dye or chemical used.Considerations which must be made in proper selection of a fadingretardant means would become clear from the following discussion.

Gill Experientia 35, 400-401 (1979) used sodium dithionite (DT) toinhibit the fading of onion cuticle cells labeled with fluorochromessuch as fluorescein, acridine orange, 33258 Hoechst, Acriflavin andothers under continuous excitation for two min with an HBO 200 W mercurybulb. It should be noted that Gill did not use these dyes conjugated toantibodies. Gill reported that for fluorescein and acridine orange, theintensity increased before starting to decrease after five min ofcontinuous excitation. Gill's data showed that after normalizing theintensities, the ratio of the intensity at two min. excitation to theinitial intensity was 0.67 for the buffer control and 1.00 for themounting medium with DT.

Giloh and Sedat Science 217, 1252-1255 (1982) incorporated n-propylgallate or 3,4,5-trihydroxybenzoic acid n-propyl ester (nPG) into themounting medium to retard the fading during serial photographs of nucleiof fixed, cultured Drosophila cells incubated with a monoclonal antibodyagainst Drosophila melanogaster embryo nuclei. It was found that 2 to 5%nPG in glycerol reduced fading of tetramethyl rhodamine isothiocyanate(TRITC) and FITC by a factor of 16 and 7 times, respectively. Atconcentrations of 10-20% nPG in glycerol, self-quenching occurred. Itwas also noted that free radical scavengers such as dithiothreitol (DTT)at concentrations of 0.05M to 0.2M in 90% glycerol had no effect onfading. Giloh and Sedat, supra suggested that the initial fluorescenceintensity may decrease upon storage in nPG. The decrease in intensitycan be reversed and prevented by washing the slides in phosphatebuffered saline (PBS) and storing them in pure glycerol.

Johnson et al., J. Immunol. Meth. 55, 231-242 (1982) addedpara-phenylenediamine (PPD) or triethlenediamine or 1,4diazabicyclo[2,2,2]octane (DABCO) to the buffered glycerol mountingmedium to reduce fading during examination of cells for ANA staining.Using a 16× Plnachromat objective and PPD at a concentration of 0.01M,they found that about 90% of the initial fluorescence intensity remainedafter 5 min. of continuous excitation with an HBO 50 W mercury lamp.DABCO provided similar protection when used at a higher concentration of0.2M. When the magnification was increased to 40×/0.95, with both PPDand DABCO, there was about 60% of the initial intensity remaining after5 min. continuous excitation. For the glycerol controls using the40×/0.95 objective, only 10-20% of the initial intensity remained after5 min continuous excitation. DABCO was recommended over PPD since thelatter is a skin sensitizer, photosensitive and undergoes oxidativedegradation.

Johnson et al., supra also compared the fading of stained nuclei in thepresence and absence of protecting agents using an HBO 50 W mercury andan HBO 100 W mercury (incident illumination) and a Quartz-Iodine (QI)lamp with transmitted, darkfield illumination. The relative initialfluorescence intensities of the three lamps were 12:5:1, respectively,for HBO 100:HBO 50:QI. It was reported that at low magnification (16×),the fading was similar for all three lamps.

An important point to note is that blank readings (unstained sectionsmounted in the same medium as the stained slide) accounted for as muchas 25% of the readings on stained cells. The blank readings weresubtracted from the corresponding reading from the stained sections. Itwas postulated that the blank reading accounted for the non-fadingcomponent described by McKay Immunology 43, 591-602 (1981). However, theblank readings that McKay used were on the stained slides from an areaof non-specific staining. The values were much lower than 25%. Whetherit is valid to use the unstained cells emission as a background for thestained cells was unclear. No data was given by Johnson et al todocument that this is a true reflection of the amount of fluorescenceintensity which the stained cells emit non-specifically. This isparticularly important since the amount was so high relative to thespecific intensity, and the counterstain was added to mask the nonspecific intensities. The counterstain emission itself is excluded bythe filter selection.

As will be further described herein infra, it has been determined thatmounting the specimen in a non-fluorescent resin reduces fading bystabilizing the macromolecule-dye complex. Rodriguez and DeinhardtVirology 12, 316-317 (1960) used polyvinyl alcohol to preparesemi-permanent mounting medium and to reduce fading upon storage. It wasreported that slides stored at 4° C. and frequently exposed to roomtemperature for hours at a time did not show appreciable fading forperiods exceeding nine months. Fukuda et al., Histochemistry 65, 269-276(1980) stained smears of mouse hepatocytes with an anti UV-DNA antibodyand FITC-labeled antibody and measured the fading in glycerin or buffeerwith post fixation with methanol. After 20 min. continuous excitation,the fluorescence was nearly immeasurable. However, post-fixation of thespecimen with absolute methanol for 1 hr followed by mounting in anon-fluorescent resin greatly reduced fading. The mechanism of thiseffect is most likely the removal of water with its component ofdissolved oxygen. This provides more rigidity to the fluorophore complexand less opportunity to interact with oxygen which accelerates thefading rate. No detectable fading was found after storing the specimens2 years at room temperature withoug shielding against light. Mckay et al(1981) found that by mounting the specimen in pure glycerol or butanolinstead of 15% glycerol, the quantum yield of RB 200 in solution couldbe doubled and the intensity of fluorescence of stained slides wasincreased by almost 50%. Again, this is consistent with the abovementioned mechanism of the decreased water concentration providing moreefficient fading protection since alcohol is a dehydrating agent.

Fukuda et al., Histochemistry 52, 119-127 (1977); Fujita et al.,Histochemistry 40, 59-67 (1974); Fukuda et al., Acta Histochem Cytochem.9, 180-192 (1976); Fujita et al., Histochemie. 36, 193-199 (1973);Fukuda et al., Acta Histochem. Cytochem. 8, 331-341 (1975) used anothermethod to eliminate fluorescence fading of the Feulgen-stained nuclei.They either pre- or post-irradiated (after nuclear staining) thespecimen for up to 20 hr to selectively remove the non-specificfluorescence and subsequently the stain retained the proportionalitybetween DNA content and stain concentration. This method is based on thefact that the non-specific fluorescence decays faster than the specificfluorescence, therefore, by carefully adjusting the pre- orpost-illumination time, one can selectively remove the unwantedfluorescence and not destroy the specific fluorescence. Fukudapost-fixed a pyrimidine-dimer FITC complex with ethanol and mounted thespecimen in Entellan (a non-fluorescent mounting resin). The specimenswere irradiated with violet light for FITC (405 nm) before or afterstaining, for five hr. They found that post-irradiation of the specimenwith violet light for appropriate times after staining reducedbackground fluorescence and decreased the fading of tissue-bound FITC.Fukuda standardized the conditions for post-irradiation for DNAcytofluorometry on a paraosaniline Feulgen stained smear and found thata post-irradiation of 10 hr retained proportionality between DNA amountand fluorescence intensity. This method essentially accelerates thefading to a plateau level and thus provides minimal subsequent fading sothat a more stable emission during measurement is obtained.

Because of the difficulty of comparing reports of the effectiveness ofprotecting agent when performed under various excitation and measurementconditions, the Applicants took the first step of testing the protectiveeffects of several chemical reducing agents under the same excitationconditions. The agents compared were sodium dithionite (DT),dithiothreitol (DTT), dithioerythritol (DTE) and triethylenediamine or1,4 diazabicyclo[2,2,2]octane (DABCO). The Applicants performed thetesting using a macrofluorophotometer for screening the effectivenessand a microfluorophotometer for verifying the "in use" conditions. Thekinetics of the fading curves were analysed and implications forelucidation of the mechanism of fading and the mechanism of theprotection were determined. The instrumentation is described first. Asis evident from the above, a standardized system or method ofdetermining and quantifying FRP has simply not heretofore been known inthe art. The Applicants now describe specific embodiments to achieve thedesired results.

DESCRIPTION OF PREFERRED EMBODIMENTS 1. Instrumentation

A Zeiss microscope-photometer is adapted for epi-fluorescence (incidentexcitation) using an HBO 100-W mercury lamp with a stabilized DC powersupply. FIG. 1 shows a schematic of the light path. A microprocessor,Zonax, is integrated with the microscope. A wide-band FITC filter set(Zeiss Product No. 487709) was used, excitation from 450 to 490 nm,dichroic mirror at 515 nm and barrier filter at 520 nm. The filters weremounted in the Zeiss III-RS illuminator filter holder which containedpositions for four combinations. A heat filter (Heat reflecting CALFLEX,Zeiss product number 467832) placed in the exciting path minimizesintensities from background materials and reduces fading. A linearinterference monochromator is placed in the emission light path to thePMT.

Attached to the monochromator on the microscope is a Hamamatsu PMT (typeR928 multi-alkali photocathode, 9 stage, side-on), powered by astabilized high voltage power supply. An amplifier was built into thePMT housing. The emitted intensity was converted into a voltagedisplayed on the computer cathode ray tube (CRT) screen. A series ofvariable field stops can mask down the area of the specimen actuallyilluminated by the exciting light. They ranged from 0.05 to 2.5 mmdiameter. Adjacent to the PMT, in the emission light path, arediaphragms that can vary the area being measured. They ranged from 0.08to 5 mm diameter. The amount of fading during the measurements can bereduced by a fast shutter which excites the specimen for milliseconds.The microscope is equipped for brightfield, darkfield and phase contrastfor localizing the specimen.

Software programs, commercially available and provided by Zeiss, controlthe microscope shutters, field stop, PMT diaphragm, high voltage, gainand the scanning stage. A measurement protocol either automatic ormanual could be employed.

Quantitative endpoint determination requires stable emitters tocalibrate the photometer unit. Suitable as stable emitters forphotometer calibration purposes are any organic or inorganic particlescoated or impregnated with any materials that are fluorescence emitters,such as UO₃, Tb³⁺, Eu³⁺ and the like.

Several types of fluorescent materials for use with microfluorometry forstandardization were evaluated. Table 6 gives the suppliers, size, shapeand the kinds of fluorescent emitters that were studied.

                                      TABLE 6                                     __________________________________________________________________________    CHARACTERISTICS OF FLUORESCENT STANDARDS                                      Name   Mfg. Material                                                                             Shape                                                                              Size     Fluorophore                                  __________________________________________________________________________    Thread NBS  Uranyl glass                                                                         Cylinder                                                                           3 × 4 × 10μ                                                             Uranium oxide                                Plate  Corning                                                                            Uranyl glass                                                                         Square                                                                             5 × 5 × 0.3 mm                                                             Uranium oxide                                                   Sheet                                                      Mount  Zeiss                                                                              Uranyl glass                                                                         Sheet                                                                              5 mm. dia.                                                                             Uranium oxide                                Phosphor                                                                             RCA  Inorganic                                                                            Irregular                                                                          Microns  Inorganic                                                elements                                                          Flurospheres                                                                         Coulter                                                                            Polystyrene                                                                          Sphere                                                                             5 or 10μ                                                                            Yellow dye                                   Covaspheres                                                                          Covalent                                                                           Latex  Sphere                                                                             1μ    Green dye                                                                     (covalent)                                   Fluoresbrite                                                                         Poly-                                                                              Latex  Sphere                                                                             0.05 to  Yellow dye                                          sciences         1μ dia.                                                                             (covalent)                                   __________________________________________________________________________

The National Bureau of Standards (NBS), Gaithersburg, MD, provided7-to-20 micron diameter threads of uranium-impregnated glass spun toachieve these small diameters; these fibers give uniform fluorescencethroughout their length. The wavelengths of the excitation and emissionpeaks are 423 nm and 534 nm, respectively. Microscope slides wereprepared by cutting the threads into fragments and using those thatapproximated the size of the bacteria of interest.

A glass filter plate (Corning Glass, Inc., Corning, NY catalog number3718) consists of glass impregnated with uranium that fluorescesyellow-green. Also, a sheet of uranyl glass, provided by Zeiss, Inc., ismounted by magnetic attachment to the objective for easy removal.

Inorganic phosphor particles including sulfides or silicates, (RCACorporation, Lancaster, PA), 1 to 10 microns, are used for cathode raytube screens. These particles can be selected to approximate theemission wavelength of the fluorophore of interest, such as FITC. Theydo not fade with continuous excitation and, when mounted dry, are stablefor many years and can be used as stable emitters.

Fluorospheres (Coulter Electronics, Inc., Haialea, FL) are polystyrenespheres that have a fluorophore incorporated into the plastic. They areavailable in 5- or 10-micron-diameter sizes. The 10-micron spheres arealso available in various fluorescent intensities ranging in arbitrarilyassigned values from 0.02 percent to 100 percent. This is achieved byadding proportionate amounts of dye during the manufacturing stage. Thewave lengths of excitation and emission are 460 nm and 550 nm,respectively. Decay is less than one percent per year if thefluorospheres are stored in a cool, dark place.

Polysciences, Inc., Warrington, PA, provides latex spheres,Fluoresbrite, in sizes ranging from 0.05 to 1.00 microns in diameter.The recommended shelf life is one to two years. These spheres can alsobe coupled to antibodies or other proteins. The excitation and emissionmaxima are 458 nm and 540 nm, respectively, for the spheres with theyellow-green dye.

Covaspheres (Covalent Technolgy Corp., San Jose, CA) are latex spheresthat are uniform in size, approximately one micron in diameter, andavailable with red or green fluorescing dyes. The user may link thesecovalently to immunoglobulins or other protein. The excitation andemission peak wavelengths for the green covaspheres are 468 nm and 537nm, respectively.

Baltimore Biological Laboratory, Cockeysville, MD, has a microscopeslide that incorporates three different inorganic phosphors into asemipermanent, non fluorescent mounting media. One phosphor emitsyellow-green light and one emits red-orange light. They do not fadeunder repeated excitation and have a shelf life of many years.

Mounting Media

All the standard materials are measured mounted in very, very lowfluorescent oil from R.P. Cargille Lab, Cedar Grove, NJ. This oil isloaded into a disposable plastic syringe fitted with a micropore filterand needle. When used for preparing slides, an amount is pushed throughthe filter onto the slide, thus ensuring a particle-free, nonfluorescent background.

For the experiment with various mounting media, Entellan (E. Merck,Darmstadt, Germany), Flo-Texx and Pro-Texx, (Lerner Laboratories,Stamford, CT), or phosphate-buffered glycerol with a pH of 8.2 was used.

Field Measurement Optimization

FIG. 2 presents the results from varying the area illuminated by theexciting light and the area sensed by the PMT. The signal-to-noise ratiois the intensity from a uranyl glass fragment approximating arepresentative bacterium area in ratio to an adjacent non fluorescentarea accounting for background. This value is plotted versus theilluminating field area and the PMT aperture area as a three-dimensionalgraph. In this experiment, a fragment of NBS thread of 3×4 microns wasmeasured. Using both 0.63-mm-diameter field and PMT diaphragms, producedthe optimal signal-to-noise ration. The projected image of thesediaphragms corresponded closest to the projected image of the uranylfragment area.

Instrument Performance

The plate was used in evaluating the reproducibility of themicroscope-photometer. Under continuous excitation, a slope of -0.008percent per second of the linear regression line and a coefficient ofvariation of 0.313 percent (Table 7) was obtained.

                  TABLE 7                                                         ______________________________________                                        Reproducibility of Fluorescent Standards                                                           VARIATION   STABILITY                                    STANDARD  INTENSITY  (CV %)      (Slope %/sec.)                               ______________________________________                                        Plate     14.95       0.3        -0.008                                       Phosphor  39.30      N.A.        -0.030                                       Fluorospheres                                                                           1.49        5.2        -0.030                                       Fluoresbrite                                                                            1.79       11.0        -0.100                                       Covaspheres                                                                             3.28       30.0        -0.300                                       ______________________________________                                    

Linearity Determination

To determine that readings were within the instrument's linear range,the intensity of a thread fragment was measured employing all usablecombinations of gain and high voltage and using the optimal field andthe PMT diaphragms. FIG. 3 shows a plot of the corrected fluorescentintensity readings of the thread versus the high voltage. Multiplyingthe voltages by the gain settings of the PMT amplifier corrected thefluorescent intensities.

Calibration Curve

To establish daily intensity readings comparisons, the fine focus of themicroscope was used to adjust the PMT output to a maximum. FIG. 4 showsthe intensity readings for the daily settings of the plate during 240hours with the same 100-W mercury lamp. Readings were taken at variousamplification settings (high voltage and gain). The regression lineparameters (slope and intercept) adjust the PMT voltage readings tocomparable values to correct for aging effects of the excitation lamp orother variables. Table 8 gives the parameters of several regressionlines for five lamps at various burn hours.

                  TABLE 8                                                         ______________________________________                                        Characteristics of Standard Calibration Curves at                             Various Burn Hours With Different Lamps.                                                        REGRESSION LINE                                             LAMP #    BURN HOURS    Slope    Intercept                                    ______________________________________                                        3         43            7.51     -19.34                                       3         330           7.54     -19.49                                       4          7            7.62     -19.69                                       4         24            7.71     -19.97                                       5         14            7.63     -19.75                                       6         100           7.51     -19.16                                       6         172           7.49     -19.33                                       7          3            7.57     -19.09                                       7         34            7.56     -19.15                                       ______________________________________                                    

Having established the performance characteristics of the instrument,various standard materials were then evaluated.

Standards Performance

The plate was tested for uniformity. The surface was scanned in30-micron increments. FIG. 5 is a typical scatter plot of thefluorescent intensity as a function of the position on the plate. Shownalso is the linear regression line, which has a slope of +0.026 percentper micron. The coefficient of variation of the mean is 0.9 percent.

FIG. 6 shows intensity from the phosphor particles under continuousexcitation. They were mounted in various mounting media on themicroscope slide. The slopes (in percent per second) of the regressionlines are +0.03 in Entellan, -0.05 in Flo-Texx, -0.18 in Protexx and-0.46 in buffered glycerol.

One drop of a 1:800 dilution of the graded fluorosphere stock materialswas added to glass slides, resulting in approximately one particle peroil immersion field (X1000). FIG. 7 gives the measured intensity valuesand the expected values based on the manufacturer's dye concentrationused for the graded brightness fluorospheres. The measurements areconsistent with the expected values measured by microscopy, whereasvalues by fluorometry deviate from the expected in several cases.

The fragments made from the NBS thread have intensities and size in therange of the bacteria expected to be of interest using these techniques.

Table 7 shows the intensity and reproducibility of some fluorescentemitters. For the last three emitters the coefficient of variation iscalculated by taking repeated millisecond exposures on 20 differentparticles. The stability is shown by the slope of the linear regressionline based on readings under continuous excitation.

Field Measurement Optimization

Optimizing the signal-to-noise ratio for the fluorescence measurement ofsmall particles on a glass microscope slide showed that the highestvalue was obtained when the area illuminated and the area sensedcorresponded to the particle area. Minimal but measurable errors fromglare or scatter in this system occurred for other size areas. Thus, theminimal area that includes the entire particle is the optimal conditionfor measurement.

Instrument Performance

Fluorescent reproducibility depends more on stability under repeatedexcitation than on instrument fluctuation. Because repeated measurementsare easily made with the semi-automated instruments, resulting inreduced variation, a less than one percent coefficient of variation canbe achieved with this system if the emitter is stable. The number ofmeasurements required varies depending on the brightness of the sampleand should be determined for each type of biological material used.

The geographical non-uniformity of each of the uranyl glass plates showsless than one percent coefficient of variation, therefore any positionon the plate can be used for calibration. Care must be exercised incleaning the plates. Glare effects necessitate avoiding the edges.

A minimal slope of the linear regression line indicates stability. Theinstrument's characteristic slope is 0.008 (Table 7); therefore, anygreater slope is presumed to indicate the emitter's fading.

Linearity Determination

The instrument response is proportional at various high voltage settingsand gains and the entire unsable instrument's range gives accuratereadings as described by a correlation coefficient of r=0.991.

Calibration Curve

Daily readings of the uranyl plate (FIG. 4) over the life of an HBO100-W mercury lamp indicate stability of the excitation source. If anyfactors are believed to affect the intensity of the sample readings,these are compensated for by mathematical adjustment based on the platereadings. This allows accuracy in making comparable readings over longperiods. Readings taken with different lamps may also be constant (Table8); however, sufficient number of lamps have not yet been used to verifythis completely. Using the characteristics (slope and intercept) of theregression lines from the calibration curves allows comparing theperformance of each lamp at various burn hours. Table 8 indicatesnegligible changes between lamps and burn hours.

Standards Performance

Each of the materials has an excitation/emission spectrum suitable foruse as a standard for FITC, although no one material has the exactexcitation/emission spectrum of FITC. The size distribution accommodatesthe range of interest for use with various species of bacteria as wellas tissue cells.

Because the geographical intensity variation is acceptable, i.e., lessthan one percent, either uranyl plate can be used daily for calibrationsof the instrumentation. This can be done without searching for the samearea on the plate, since any area gives the same intensity within onepercent. Therefore, voltage reading at any random plate location isadjusted to maximum intensity with the microscope focus. Thiscompensates for the effects of aging on the excitation lamp and of othervariables.

The graded brightness fluorospheres are useful in checking the linearityof the measurement system and in selecting a standard of the requiredbrightness within the range of interest. The spheres are convenient tohandle, and slides can be prepared with one particle per field. Thisallows testing the effects of glare on the intensity measurements.

The daily fluctuations of the uranyl fragment are small. Therefore thistype of standard is stable and also suitable for compensating for theaging of the lamp. However, it is more tedious to locate the fragmentthan focus on the plate.

The emitters have excitation and emission spectra compatible with mosttypes of instrumentation that are used for FITC measurements.

The phosphor particles are irregular in size, and therefore nocomparison of individual intensities for use as fluorescence sourcestandards is meaningful.

A larger variation in fluorescent intensity occurs with the covaspheresand the fluoresbrites. These particles are much smaller than others,which may account for the difficulty in achieving less variability.

The slope of the intensity measurements of the plate, phosphors andfluorospheres are within the variation of the instrumentation, thereforefading is not considered measureable with these emitters. Thecovaspheres and fluoresbrite show a degree of fading that may bebothersome only in some applications. Using a narrower band pass filter(440-490 nm) results in reduced fading of covaspheres, i.e., a slope of0.10 percent per second. This filter may be used with FITC-containingmaterials. The covaspheres and fluoresbrite do provide a particle thatcan be covalently coupled with a specific antibody. Further, they mayprovide a model for the fading of fluorophores.

In summary, of the types of calibrators considered, each is useful fordifferent purposes. The uranyl glass materials have the greateststability, being stable under continuous excitation and can be used toset values for the instrument. The inorganic phosphors are also suitablefor calibration since they are stable with continuous excitation. Theirirregular size however, causes intensity variations and thus can only beused if particular particles are selected. The fluorospheres areavailable in graded intensities and sizes and can be used to approximatebacteria type particles. The covaspheres and fluoresbrites can be bondedto an antibody, the latter being available in the smaller size range.

Using the uranyl glass plate or the magnetically mounted plate allowscomparison of daily intensities produced by the microscope system andaccounts for effects from the lamp and its aging. Use of regression lineparameters allows monitoring variability.

Consistency can thus be obtained in values for fluorescent intensitymeasurements that enables interlaboratory comparisons andstandardization.

As described herein supra, one of the most reliable of the calibrationstandard was the uranyl plate (Corning, Product No. 3718). By specialorder, a modification of this uranyl plate was provided in the shape ofa microscope slide. The slide was found to be non-fading and thus wasused to evaluate the stability of the microscope-photometer. Undercontinuous excitation for three hours, a negligible slope of -0.008percent per second of the linear regression line and a coefficient ofvariation of 0.313 percent was obtained.

Using the uranyl glass slides with the optimal field stop and PMTdiaphragms, the fluorescence intensity values were determined using allpossible combinations of amplifier gain and high voltage settings whichresult in measureable intensities. Multiplying the readings by the gainsettings of the PMT amplifier for each high voltage setting correctedthe readings for various gain settings. The corrected intensity-voltagerelationship was linear on a log-log plot. This indicates that theintensity is related to the high voltage as a power function, as isexpected. The equation for this was log (PMT output)=(7.5) X log (highvoltage)-19.34.

To compare intensity readings day-to-day, the uranyl glass slides wereread before and/or after each experiment. Since the uranyl slidescontain nothing upon which to focus, the focus knob was adjusted unilthe highest intensity reading was obtained. The maximum intensity wasconstant across approximately one half of a turn of the fine focus knob,indicating that the focal level on the uranyl glass slide is notcritical within this amount and allowing confidence in the readings.Since the uranyl slide contains fluorophore throughout its entirethickness, it is assumed that the focal level of maximum intensityrepresents where the focal cone is filled with fluorescence from a solidangle relative to the numerical aperture of the objective used. Analternative method has been suggested, to scratch with a diamond point amark on the surface of the glass and to focus on the mark. This however,is not satisfactory for two reasons: the scratch is hard to find andoften disappears when oil of certain refractive index is added to theslide; also, the focal level is at the surface of the slide and sincethere is no fluorophore above the surface, small variations in focuswill introduce large variations in the amount of light measured. Theinitial reading of the uranyl glass slide was set to 100 for ease ofmathematical manipulation and to make full use of the graphics screen onthe CRT. The high voltage was 517 volts and the amplifier gain was one.All further readings of both standards and samples were made at highvoltage and gain settings that gave relative intensity values close to100. Then these high voltage and gain values were used to correct theintensity readings of the sample relative to the uranyl glass slidereading for the day. This was done using the regression line parametersto calculate the extrapolated intensity at the standard settings of 517and one.

c. Perkin Elmer calibration

The Perkin-Elmer 650-40 Spectrophotofluorometer ismicroprocessor-controlled and includes software to correct the samplefluorescence spectrum by reference to the emission spectrum of RB 200.This is actuated by setting the corrected mode after running the RB 200spectrum. The fluorometer uses a second photodiode to automaticallycorrect the dynode voltage for fluctuations caused by the light sourcewhich is a 150 W Xenon lamp with stabilized power supply. This isactuated by setting the ratio mode. The fluorescence intensity readingsare displayed in digital form on the fluorometer display. Anothersoftware option allows repeated scanning of the fluorescent specimenbetween pre-selected wavelengths and an average curve to be drawn fromthe individual curves.

2. Reagents

a. Reducing agents

The chemical reducing agents tested were: sodium dithionite (DT, sodiumhydrosulfite), Aldrich Chemical Company, Milwaukee, Wis., catalog#15,795-3, dithiothreitol (DTT), Sigma Chemical Company, St. Louis, Mo.,catalog #D0632; dithioerythritol (DTE), Sigma Chemical Company, catalog#D8255; DABCO (1,4 Diazabicyclo [2.2.2]Octane), Aldrich Chemical Company(catlog #D2,780-2) and n-propyl gallate, Sigma Chemical Company (catalog#P3130).

DT, DTT and DTE were prepared as stock solutions containing 0.5Mreducing agent in 0.5M TRIS buffer, pH 8.2, (Trizma base, Sigma ChemicalCompany, product #T-1503). Stock solutions were aliquoted and frozen forfuture use to preserve the potency of this material. For use in thePerkin Elmer macrofluorophotometer, doubling dilutions of the reducingagents were prepared in the concentration range 0.5M to 0.063M in 0.05MTRIS, pH 8.2. The dilution reducing agent was then diluted 1 partreducing agent to 9 parts of a mixture of 0.05M TRIS pH 8.2 andFITC-labeled conjugate. For experiments in the Zonax microscope,dilutions in the range 0.25 to 0.5M were prepared. One part of theconcentrated reducing agent was added to 9 parts buffered glycerolmounting medium.

For DABCO, doubling dilutions at a concentration range of 0.03 to 0.5Mwere prepared in 0.5M TRIS, pH 8.2 for determination of the optimalconcentration and a final concentration of 0.3M in buffered glycerol wasobtained by diluting a stock solution for other experiments.

b. Conjugates

The following FITC-labelled conjugates were used: Goat anti-humanpolyvalent globulin to Rubella virus with rhodamine counterstainincorporated (ENI, Columbia, Md.); Goat anti-human polyvalent globulinwith rhodamine counterstain incorporated for ANA ENI); Goat anti-humanIgG (heavy and light chains) with Evans blue counterstain for Toxoplasmagondii (ENI); Goat anti-human polyvalent globulin without counterstainincorporated (Centers for Disease Control, (CDC), Atlanta, Ga.) for T.gondii; Neisseria gonorrhoeae rabbit anti-human (IgG) globulin FITCconjugate with and without rhodamine counterstain.

3. Methods

a. Measurement of fading in macrofluorophotometer

The fading of FITC-labeled conjugates (without added cells) with varyingconcentration of reducing agent present was measured in the Perkin-Elmerfluorometer and compared to controls. The following conjugates weremeasured: T. gondii (without counterstain) and N. gonorrhoeae (with andwithout rhodamine counterstain). The excitation and emission wavelengthsused were, respectively, 498-nm and 522-nm. The slit widths forexcitation and emission, respectively, were 20-nm and 5-nm. The sampleswere continuously excited with a 150-W Xenon light for 10 min andintensities integrated for 15 sec intervals using the corrected spectrumoption and a plot of corrected intensity versus time was prepared.

b. Measurement of fading in microfluorophotometer.

Measurement of the fading in the Zonax microscope was done on the kitsfor ANA, Rubella and Toxoplasma. The IF microscopy slides fromcommercially available kits were prepared according to eachmanufacturer's directions except that an optimum concentration ofreducing agent was incorporated into the buffered glycerol mountingmedium provided with the kit just prior to mounting the slides. Wheneverpossible, the cells were located under transmitted visible light inorder not to allow fading of the specimen. The high voltage and theamplifier gain to the PMT were adjusted so that the initial intensitywould be 100%. The sample was continuously exposed to excitation lightusing a wide-band FITC filter combination (Zeiss product No. 487709) andintensity measurements were automatically taken every 0.1115 min for 10min. using the kinetics software. A KP-560 bandpass filter was placed inthe emission path to eliminate red emission light. The fluorescenceintensities were later corrected to a standard high voltage and gain,based on the statistical regression parameters of the uranyl glass slideused to calibrate the instrument daily, to allow direct comparison ofcell intensities independent of high voltate and gain settings and dailylamp fluctuations. Background readings were taken using the same filtercombination as for the samples by measuring an adjacent area of thestained tissue or cells that showed the non-specific staining using thesame diaphragm areas. The background readings were usually less than 1%of the sample readings. The background readings were subtracted from thereadings of the specific intensities.

c. Statistical Analyses

One of the software packages available with the Zonax allows thegeneration of kinetics graphs (a graph of intenstiy over auser-predetermined time frame). The kinetics plot allows one to lookdirectly at the percent fading of the sample. The software alsocalculates the coefficient of variation (CV%) which allows comparison ofthe fading of the cells independent of the mean. Use of a data linkbetween the Zonax and an IBM host computer allowed generation of avariety of statistical analyses including regression, analysis ofvariance and graphics output from the original data generated by themicroscope.

C. Results 1. Selection criteria

DT, DTT, DTE and DABCO were tested to determine if they couldeffectively protect the FITC-labeled cells from fading. These agentswere evaluated on the basis of five criteria:

a. Effective protection of the sample from fading.

b. No inhibition of the initial fluorescence intensity of thefluorophore.

c. No increase in the background fluorescence.

d. Able to function with the buffer, pH, molarity and temperature usedwith the mounting medium in the fluorescence test kits.

e. Practical to use.

The Perkin-Elmer Spectrofluorophotometer was used to screen the reducingagents for their protective ability. The fluorometer has the advantageof allowing rapid screening of the prospective agents without requiringseveral hours to prepare IF microscopy slides

2. Buffer type

Any suitable buffer capable of buffering in the pH range of about 6.6 to9.4, preferably in the pH range of about 7.5 to 8.4 can be used. Glycineadequately buffers in this range but has a high background intensity(autofluorescence). TRIS buffer maintains the pH within the desiredrange and does not significantly autofluoresce at the excitation andemission wavelengths used. PBS did not adequately buffer the reducingagent solution and was, therefore, not used further.

3. Buffer concentration

Experiments were performed to determine the lowest concentration of TRISbuffer that was still capable of holding the pH of the reducing agent atabout 8.2. A 2.0M stock solution of DTT was diluted in TRIS buffersolution ranging from 0.2M to 0.02M and the pH of the solution wasmeasured. 0.05M TRIS was the lowest concentration of TRIS capable ofmaintaining a pH of 8.2 (see Table 9).

                  TABLE 9                                                         ______________________________________                                        OPTIMIZATION OF BUFFER CONCENTRATION                                          FOR DITHIOTHREITOL                                                            Tris (hydroxymethyl)                                                          aminomethane                                                                  Concentration 0.20 M  0.10 M    0.05 M                                                                              0.02 M                                  ______________________________________                                        pH                                                                            with 0.2 M DTT                                                                               8.38    8.24      8.14  8.03                                   without DTT    8.38    8.25      8.21  8.11                                   Background Intensity                                                          (Relative Light Units)                                                        with DTT      15.93   10.83     10.72 10.66                                   without DTT   18.77   14.19     12.57 10.20                                   ______________________________________                                    

4. Reducing agent concentration

Various concentrations of reducing agents were added to a constantvolume of rehydrated FITC-labeled conjugates. The percent remainingafter 30 min excitation (readings taken every min) in the Perkin Elmeris shown in FIG. 8. Data are shown for DABCO with Rubella antibody andDTT with Toxoplasma antibody. DTT or DTE shows the most protection ofthe agents tested when used at their optimal concentration of 0.033M. Itis noted that the optimal concentration for DABCO is 0.3M, which is thesame as recommended by Johnson, et al. 1982. This is a concentrationwhich is ten times higher than that used for the other agents. Table 10compares the fading of the FITC-conjugate after 10 min continuousexcitation at the optimal concentrtion for each of the reducing agentsDABCO, DTT and DTE. The error around each measurement was less than 1%and does not show up on the graphs. A 10 min measuring period was chosenover the previously used 30 min period in order to make the times morecompatible with those used in the microscope and those that might beused in a clinical laboratory.

                  TABLE 10                                                        ______________________________________                                        SELECTION OF OPTIMAL REDUCING AGENT IN                                        MACROFLUOROPHOTOMETER                                                                     DTE      DABCO    Unprotected                                     ______________________________________                                        Background Intensity                                                                        25.5*      25.7     5.7                                         in 0.05 M TRIS                                                                Optimum concentration                                                                       0.033 M    0.3 M    not appl.                                   in 0.05 M TRIS                                                                Conjugate:                                                                    Initial intensity                                                                           1049.3*    701.5    1049.3                                      minus background                                                              10 min intensity                                                                            918.4      654.5    726.3                                       minus background                                                              % Remaining intensity                                                                       87.8%      94.0%    69.0%                                       based on initial                                                              intensity                                                                     % Remaining intensity                                                                       87.8%      64.5%    69.0%                                       based on unprotected                                                          initial intensity                                                             ______________________________________                                         *Relative Intensity Units                                                

5. Microscopic verification of protection

After suitable protecting agents were found by screening in themacrofluorophotometer, the protective ability of these reagents wasverified by incorporating the reducing agents into the mounting mediumof the IF microscopy slides.

FIG. 9 is a plot of percent remaining intensity after 10-min continuousexposure to excitation light, with and without reducing agent present.Shown are results with Toxoplasma, Rubella ana ANA test kit slidesmounted in kit buffered glycerol with or without 0.025M DTE added. Sincethe labeled specimen fade too rapidly to record the initial, unfadedintensity, the first possible intensity (within each kit) which is thereading with the highest intensity, is used as the initial intensity. Itshould be noted that in the case of ANA and Rubella, unprotected, only 8and 5%, respectively, of the protected intensity remained after 0.1 min.The initial intensity with DTE was 10 times greater than the unprotectedintensity. It may also be noted that when visually observing the cellswithout DTE, the cells were totally red (due to counterstain) after 1min continuous excitation. For cells with DTE, after 10 min continuousexcitation, the cells were still fluorescing brightly green. Datasimilar to that shown in FIG. 9 was obtained using DT. However, due tothe inability of TRIS buffer to maintain a pH of 8.2 in the DTsolutions, this reducing agent was not used in future experiments.

Verification of the effectiveness of the selected concentrations of thereducing agents on FITC-labeled conjugate to ANA with rhodaminecounterstain-treated cells during continuous excitation in the Zonaxmicrofluorophotometer microscope was made. A comparison of percentremaining intensity with 0.025M DTE, 0.3M DABCO and no reducing agent inthe buffered glycerol for 10 min continuous excitation is shown in FIG.10. It should be noted that after 0.1 min, only 20% of the initialfluorescence intensity remains for DABCO. Even after 10 minutescontinuous excitation, 20% of the initial intensity remains for DTE ascompared to 2.2 and 1.3% for DABCO and buffered glycerol, respectively.However, if DABCO is compared with its own initial intensity as 100%,then, the protection appears to be effective at retaining 25% of its owninitial intensity. This indicates that the fading is accelerated by theDABCO initially and then changes at a slower rate and thus impartsstability to the subsequent readings. It may, therefore, be of value asa protector when used for viewing the samples repeatedly. DTE waschoosen for subsequent use on the basis of its higher initialfluorescence intensity and greater protective ability over the 10 minmeasuring period of samples containing DTE, although DTT was almost aseffective.

Efforts were made to improve the protective ability DTE without loweringthe initial fluorescence intensity. FIG. 11 shows the fading protectionwhen 0.025, 0.033 or 0.05M DTE was incorporated into the mounting mediumfor the ANA IF microscopy kit. It should be noted that there is almost a40% lowering of the initial intensity with 0.05M DTE. Also, 0.05M DTEdoes not protect the sample better than 0.033M during the first 3.5 minof excitation. Therefore, 0.033M DTE is chosen due to the higher initialfluorescence intensity. The time required to align the specimen in theexcitation field is normally less than two minutes, during which 0.033DTE retains its protective ability.

6. Selection of reducing agent

Base on the above-stated criteria, DTT and DTE were selected as thepreferred protecting agents although others could also be used. Thesereagents offered the most protection from fading with the leastinhibition of the initial intensity. It is noted that DABCO at 0.3Mlowered the initial intensity 23%.

D. Conclusions 1. Optimization of chemical environment

The data presented herein indicate that the fading behavior of cell-freeconjugates in the fluorometer is a good predictor of the behavior of thefluorophore in the microscope-photometer.

a. Buffer selection

Since the intensity of the fluorescence is dependent on the pH of themedium, it is very important to choose a buffer that can maintain the pHat an optimum value of about 8.2 to 8.5. After testing several buffers,including glycine, TRIS and carbonate, TRIS buffer was found to hold thepH in the optimum range in the presence of reducing agent and also tohave the lowest background intensity.

b. Reducing agent selection

The ultimate usefulness of the reducing agents is to inhibit fadingwhile the specimen is being examined microscopically. Therefore, thereducing agents must be able to function with the buffer and pH of themounting medium. DTE was selected as a preferred reducing agent basedupon the selection criteria listed supra. DTE significantly reduced thefading over a 10 min continuous excitation period and did not reduce theinitial intensity of the fluorophore. In addition, DTE is easy andpractical to use with the buffered glycerol mounting medium. Based onthe results in FIG. 8 with DABCO, it is possible that if it were used ata concentration of 0.1M or less, one would encounter only a 10% increasein fading, while the initial intensity would be much higher since higherconcentrations of DABCO suppress the fluorescence intensity.

c. Reducing agent concentration

It is necessary to find the lowest concentration of reducing agent thatis able to prevent fading for several reasons. First, if theconcentration of the agent were too high, reduction of the initialfluorescence may occur and possibly cause faulty end-pointdetermination. Second, the concentration must be low enough so thatbackground intensity is not increased due to autofluorescence of thereducing agent or precipitation of salts on the slide. Table 11 givesthe final reducing agent concentration and pH which are preferred inaccordance with the present invention for incorporating DTE intobuffered glycerol.

                  TABLE 11                                                        ______________________________________                                        MOUNTING MEDIUM FOR OPTIMAL PROTECTION                                        Final Concentration in Buffered Glycerol                                      ______________________________________                                        TRIS concentration    0.05    M                                               DTE concentration     0.033   M                                               pH                    8.0-8.2                                                 ______________________________________                                    

2. Kinetics

A more negative slope (%/sec) of the linear regression curve is ameasure of fading. Comparing the slopes of the linear regression linesfor samples with different reducing agents allows a quick and precisemethod of comparing the rate of decrease in intensity over a given timeperiod (Table 12). DABCO shows a more negative value and thus indicatesgreater fading. The other agents show similar slopes and thus areequivalent in protective function when used at their own optimalconcentration.

                  TABLE 12                                                        ______________________________________                                        SLOPE OF FADING WITH DIFFERENT                                                REDUCING AGENTS                                                                                      SLOPE                                                  AGENT         conc. M  % per sec.                                             ______________________________________                                        DABCO         0.3      -17.02                                                 DT            0.25     -4.64                                                  DTT           0.25     -4.30                                                  DTE           0.25     -4.18                                                  ______________________________________                                    

FIG. 12 shows the change in fluorescence intensity as a function ofexcitation time of the labeled Rubella cells mounted in bufferedglycerol or with DTE added. When DTT or DTE are present the best fit isa linear regression curve, whereas with no protection, the best fit is aquadratic regression curve. This means that with the reducing agentpresent, the reaction is of first order, or a function of one ratelimiting factor, and without it, the reaction is due to the interactionof two rate limiting factors. This difference may represent twodifferent mechanisms of fading, an oxygen sensitive mechanism and anon-oxygen sensitive mechanism (FIG. 13). Without being bound to anytheory, it is hypothesized that the fading in the presence of thereducing agent is non-oxygen sensitive. It should be pointed out,however, that the reducing agents only scavenge the oxygen and that, inno way in the studies described herein, have exhaustive measures beentaken to completely remove oxygen from the mounting medium.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application and thescope of the appended claims.

We claim:
 1. A method for quantitative determination of fluorescentendpoint of a fluorescent reaction in photometric microscopycomprising:(a) incorporating a protective agent in a mounting medium forfluorescent reaction in an amount sufficient to reduce fading offluorescent reaction product less than 25% of initial fluorescentintensity; (b) calibrating the photometer used in said microscopy with astable emitter; and (c) recording the intensity of fluorescence of saidfluorescent reaction product by means for measuring light intensity. 2.The method of claim 1, wherein said protective agent is selected fromthe group consisting of sodium dithionite, dithioerythritol,dithiothreitol and triethylenediamine.
 3. The method of claim 2 whereinsaid protective agent is dithioerythritol.
 4. The method of claim 2wherein the amount of said protective agent in said mounting medium isin the range of about 0.01M to about 0.3M.
 5. The method of claim 4wherein said mounting medium comprises a suitable buffered medium havinga pH value ranging from about 6.6 to about 9.4.
 6. The method of claim 5wherein said pH value range from about 7.5 to about 8.4.
 7. The methodof claim 6 wherein said buffered medium comprises about 0.05M glycine ortrizma base.
 8. The method of claim 7 wherein said buffered mediumcomprises about 0.05M trizma base.
 9. The method of claim 1 wherein saidstable emitter is selected from the group consisting of organic andinorganic particles coated or impregnated with any material that arefluorescence emitters.
 10. The method of claim 9 wherein said emitter isuranium impregnated glass, fluorescently labelled polystyrene spheres,fluorescently labelled latex spheres, inorganic ions and inorganicphosphor particles.
 11. The method of claim 10 wherein said emitter isuranyl glass plate.
 12. The method of claim 1 wherein said means formeasuring light intensity is a computer controlled microscope voltageoutput photometer.